1. Field of the Invention
This invention is in the field of avian coccidiosis and is related to a DNA molecule encoding an antigenic protein of Eimeria apical membrane antigen 1 and use thereof.
Coccidiosis is an intestinal disorder of poultry and causes an assortment of problems in the infected host. These problems range from poor feed conversion ratios in light infections to acute death in heavier infections.
Coccidiosis is caused by protozoans belonging to the genus Eimeria. The members of this genus in poultry are E. acervulina, E. tenella, E. maxima, E. necatrix, E. brunetti, E. mitis and E. praecox. Some investigators include E. mivati and E. hagani into the member. All of these species have similar life cycles but display different tissue specificity and pathogenicity. A broiler chicken will be subjected to a great deal of damage by E. acervulina or E. maxima because they parasitize large portions of the small intestine, where food digestion plays a major role.
Coccidiosis can be controlled by the administration of anti-coccidial agents. However, drug resistant strains arise at a frequent rate and the cost of development of new drugs is quite high. In addition, a number of these agents leave residues in the meat, which might give problems on consumer.
Attempts have been made to prevent the disease by vaccinating chickens with live attenuated strains of Eimeria or inactivated parasites. These live attenuated strains such as precocious lines are obtained by inoculating chickens with oocysts of a wild Eimeria species and collecting the very first parasite that are excreted as a result of infection (J Parasitol. 1975, 61: 1083-1090). However, such attenuated live vaccines produce fewer parasites and give an appreciable disease effect to vaccinated chickens. On the other hand, a protection level using the latter (inactivated vaccine) is far from complete. Furthermore, the disadvantage of these vaccines is expensive to be produced because a large-scale production of these vaccines needs a lot of live chickens.
An alternative solution would be to produce, by genetic engineering, the protective antigens of Eimeria parasites. Once developed, these immunogens could be produced cheaply in a prokaryotic or eukaryotic culture system in an unlimited supply and used to vaccinate chickens against coccidiosis.
2. Related Art
Several protective antigen genes of Eimeria have been reported. For examples, Jenkins et al. reported screening using a rabbit serum against the membrane fraction of E. acervulina, and a part of the cDNA encoding a 250-kDa protein in parasite surface (Exp. Parasitol. 1988; 66: 96-107, U.S. Pat. No. 5,122,471). Some Eimeria antigen genes were screened using monoclonal antibodies to Eimeria parasites instead of antisera (U.S. Pat. Nos. 5,028,694, 5,279,960, 5,814,320, 5,449,768). However, these antigens could elicit only partial protection against Eimeria infection to chickens immunized with a recombinant protein or recombinant virus expressing the antigen (U.S. Pat. Nos. 5,387,414, 5,403,581, 5,602,033, 6,001,363).
Plasmodium that causes human malaria is closely related to Eimeria, and both parasites belong to the phylum of Apicomplexa. Malaria is one of three major infection diseases of human, and there are an estimated 500 million infected persons, with 1-2 million dying annually. Therefore, many research teams focus on developing malaria vaccine to control infection. Plasmodium apical membrane antigen 1 is currently, one of the most promising antigens for Malaria vaccine (Molecular Microbiology (2004) 52, 159-168, The Journal of Immunology 172 (2004) 6167-6174, and Infect. Immun. 72 (2004) 4464-4470). The Eimeria apical membrane antigen 1 (AMA-1) gene is expressed only small amount in the specific organ of Eimeria. Therefore the purification of the antigen was difficult, and full amino acid sequence was unknown. Because there was no purified AMA-1, it was difficult to get the AMA-1 gene by the general screening method that is to obtain an antibody against the antigen and to screen a cDNA library with the antibody.
As described above, there is no satisfied vaccine against Avian Coccidiosis. In addition that, a DNA molecule encoding Eimeria AMA-1 protein has not been cloned and known in terms of usefulness for vaccine antigen.